The cell cycle seems to be controlled by a molecular machine that generates and regulates its own oscillations. At the heart of this machine is a ubiquitous cyclin-dependent protein kinase. In the yeast S. cerevisiae, the catalytic subunit of the kinase is the Cdc28 protein, and the regulatory subunit is one of at least nine different cyclins, Cln1 to 3 (which regulate the G1/S transition), and Cln1 to 6 (which regulate S, G2, and M). Different ClnCdc28 or Clb-Cdc28 complexes regulate different cell cycle events by phosphorylating unknown substrates. The goal of this project is to find some of the relevant in vivo substrates of the Cdc28 kinase to help us understand mitosis at the molecular level. The first part of the project will be to define the phosphorylation sites of the mitotic Cdc28 kinase complexes using synthetic peptides, and otherwise characterize their biochemical activities. It may be that all the different Clb-Cdc28 complexes will have the same or similar site specificities; in this case we will define this specificity. Alternatively, the different Clb subunits may alter the site specificity of Cdc28; this possibility will be examined. The second part of the project will be to identify substrates. We have already found a set of yeast proteins that have critical cell cycle functions and clusters of potential Cdc28 phosphorylation sites, and we believe that this set is likely to include many real substrates. Potential Cdc28 phosphorylation sites in these candidates will be destroyed by in vitro mutagenesis to see if these mutations cause a phenotype. Initial mutations will be made according to our current ideas of Cdc28 sites. Later, when better information about Cdc28 sites becomes available from the first part of the project, this will be used to guide mutagenesis. At the same time, candidate substrates will be examined for cell-cycle regulated, CDC28- dependent phosphorylation, and we will ask whether they can be phosphorylated by Cdc28 in vitro. This multi-disciplinary approach should identify a number of relevant in vivo substrates, which will help us understand mitosis and the rest of the cell cycle. The cell cycle of yeast and mammals show remarkable similarity, so these studies should be relevant to humans. Many medical problems such as cancer, aging, and wound healing are cell cycle problems, and may be better managed when we understand the fundamental mechanisms involved.